Fatty acid antimicrobial

ABSTRACT

The novel antimicrobial composition is disclosed including from about 60 to 95% of a lipophilic polar solvent such as propylene glycol, ethylene glycol, or isopropanol and from about 0.5 to 5% of a mixture of C 8  to C 14  fatty acids. Other constituents of the present invention may include water, an alcohol (such as ethanol or propanol) or a mixture thereof. Also disclosed is a novel method of killing harmful microbes on the udder of a milk-producing animal using the above-mentioned composition.

[0001] This application is a continuation of application Ser. No.08/602,498 filed Feb. 20, 1996, the disclosure of which is incorporatedby reference herein.

FIELD AND BACKGROUND OF THE INVENTION

[0002] This invention is directed to an antimicrobial composition, andin particular to an antimicrobial composition including a mixture offatty acids of differing lengths and a lipophilic polar solvent.

BACKGROUND OF THE INVENTION

[0003] The treatment and prevention of mastitis in dairy cows continuesto be of primary importance to those engaged in the dairy farmingindustry. The combined costs of mastitis to the U.S. dairy farmingindustry have been estimated at between two and three billion dollarsannually.

[0004] Mastitis is caused by infections of the mammary, ormilk-producing, glands by a broad spectrum of pathongenicmicroorganisims such as Staphylococcus aureus, Streptococcus agalactiae,Escherichia coli, Mycoplasma bovis and Candida albicans. In particular,when the milk-producing glands and surrounding tissues in the udderbecome infected, the tissues become inflamed with cellular infiltratesand associated toxic substances.

[0005] The cellular infiltrates and associated toxins, along with theinfecting organisms themselves, can cause a dramatic reduction in thequality of milk produced by the animal. The infiltrates, toxins, andorganisms can also affect the quantity of milk produced by the animal,possibly even resulting in the stoppage of production.

[0006] Occasionally, the infection can spread systemically to otherorgan and tissue sites via the blood or lymphatic systems. The spreadinginfection can, in extreme cases, seriously debilitate or kill theinfected animal.

[0007] Given the importance of the mastitis problem to dairy farmers,several methods have been proposed to combat this menace. One methodfrequently used to combat the problem has been to “cull” out or separatethe infected animals from the herd, and then to treat the infectedanimals with antibiotics. Antibiotics can be administered eitherdirectly (via an injection) or indirectly (via feed).

[0008] The secondary problem of antibiotic residues in the treatedanimal and their milk products, however, has come under increasedscrutiny from federal and state regulatory agencies, such as the UnitedStates Department of Agriculture and the Food and Drug Administration.Additionally, public outcry over the use of antibiotics and the presenceof antibiotics residues in meat and milk products has severely limitedthe market for such products.

[0009] As an alternative to treatment with antibiotics after infection,products have been designed to prevent mastitis by killing thepathogenic organisms which might otherwise infect the teat and uddertissues before the organisms enter the tissues. One such proactiveproduct is a topical antiseptic commonly known as teat (or udder) dip,wash, spray, or wipe. This product is applied to the teat and udder areaof the dairy cow or other milk-producing animal before and/or aftermilking as part of a process of general dairy hygiene. The product isintended to kill or reduce in number the mastitis-causing microorganismson the surface of the teat before the microorganisms have had a chanceto migrate or be propelled (during milking) into the teat canal, or toenter the teat via injuries or lesions.

[0010] Although the wide-spread use of topical antiseptics in the last30 years has greatly decreased the incidence of mastitis, many of theproducts presently in use as teat dips, washes, sprays or wipes(broad-spectrum chemical germicides such as chlorinated compounds,iodophors or chlorhexidines) are known to irritate the animals' skin.This is particularly significant because the cow is subjected torepeated applications of the product, two or three times a day, beforeand/or after milking, for a period of years. In some cases, thesegermicides have actually been found to be toxic to the animals and tothe human dairy workers.

[0011] Additionally, there is growing concern among the federal andstate regulatory agencies, such as the Food and Drug Administration,about the presence of germicide residues, such as iodine orchlorhexidine, in milk products.

[0012] Furthermore, chemical germicides such as chlorine, iodine andchlorhexidine compounds lack a high degree of stability. These chemicalgermicides can be become inactivated over time, or can becomeinactivated by substances (such as water or organic materials) which maycontaminate or dilute the germicide after it has been applied to theteat.

[0013] The lack of stability is a particularly significant disadvantageconsidering that, in some applications, teat dips, washes, sprays, andwipes are intended to remain on the teat and udder for hours at a timeso as to provide extended protection from pathogenic microbes betweenmilkings. In fact, for compounds such as chlorine dioxide, the lack ofstability over time becomes even more significant in that the timebetween the preparation of the product by the farmer and the applicationof the product to the animal may be at least two or three hours. Thelack of stability over time also negatively impacts the ability of thedairy farmer to store, for example, compositions made of chloridedioxide for use at a later date.

[0014] One suggested substitute for the chlorinated compounds,iodophors, and chlorhexidines presently used as teat dips are the fattyacids and their derivatives. The antimicrobial or germicidal propertiesof short to medium-chain fatty acids (C₆ to C₁₄) and their derivatives(such as esters) have been widely known for some time. See U.S. Pat. No.4,406,884; Fawzi and U.S. Pat. No. 5,208,257 to Kabara; Viegas, et al.,Inhibition of Yeast Growth by Octanoic and Decanoic Acids Producedduring Ethanolic Fermentation, Applied and Environmental Microbiology,January 1989; J. J. Kabara, Toxicological, Bactericidal and FungicidalProperties of Fatty Acids and Some Derivatives, Journal of American OilChemists' Society, November 1979; J. Fay and R. Farias, InhibitoryAction of a Non-Metabolizable Fatty Acid on the Growth of Escherichiacoli: Role of Metabolism and Outer Membrane Integrity, Journal ofBacteriology, December 1977; and J. J. Kabara, Antimicrobial Lipids:Natural and Synthetic Fatty Acids and Monoglycerides, Lipids, March1977. Fatty acids have been included in the class of lipophilic weakacids which are generally considered to be an important class ofantimicrobial agents. See Thomas R. Corner, Synergism in the Inhibitionof Bacillus subtilin by Combinations of Lipophilic Weak Acids and FattyAlcohols, Journal of Antimicrobial Agents and Chemotherapy, pp. 1082-85(June 1981).

[0015] While highly bactericidal, undiluted fatty acids are irritatingto the skin, and may even be corrosive. Fortunately, it has been foundthat dilute concentrations of fatty acids have antimicrobial efficacy.Hence, a significant amount of work has been done to prepareantimicrobial compositions using a fatty acid diluted, for example, withwater.

[0016] Preparing such a composition diluted with water is complicatedbecause short to medium-chain fatty acids are, at best, only slightlysoluble in water. One solution to the relative insolubility of fattyacids has been to add hydrotropes to compositions containing lowconcentrations (0.1 to 5.0% by weight) of a mixture of fatty acids (C₆to C₁₂) to solubilize the fatty acids. In such a composition, theshorter-chained fatty acids (C₆ to C₉) may actually assist the action ofthe hydrotrope by helping to solubilize the longer species, and therebyimproving the longer species' antimicrobial efficacy. See U.S. Pat. No.4,404,040 to Wang et al.

[0017] To maintain the antimicrobial action of the fatty acids insolution with water, however, the pH of the composition must besufficiently low (below 4.0) to allow the acids to remain in theiractive free acid form. A strong organic or inorganic acid must be addedto lower the pH so that the fatty acid can remain in its active form.

[0018] Alternatively, while teaching a fatty acid composition dilutedwith water, U.S. Pat. No. 4,406,884 to Fawzi et al. teaches away fromsolubilizing the fatty acids in water. Instead, Fawzi teaches that theantimicrobial efficacy of the fatty acids may be enhanced bysupersaturating the aqueous phase of an aqueous lotion or gel with lowconcentrations of a mixture of short and medium-chain fatty acids.According to Fawzi, the supersaturated aqueous phase combined with thelipophilicity of the fatty acids provides the increased antimicrobialaction, without resort to either a hydrotrope or solubilizer to maintainthe fatty acids in solution with the water.

[0019] Fatty acids have been found to be more soluble in vehicles otherthan water. In fact, while not specifically suggested as anantimicrobial, it has been disclosed that high concentrations (5% byweight or more) of a single length of fatty acid (C₁₀, for example) insolution with ethanol or propylene glycol may have some efficacy in thetreatment of skin diseases, such as erythroquamose and papulose. SeeGerman Laid-Open Application No. 2,912,438 to Eckert et al. SUMMARY OFTHE INVENTION

[0020] A novel antimicrobial composition is disclosed including fromabout 60 to 95% of a lipophilic polar solvent, such as propylene glycol,ethylene glycol, glycerol, or isopropanol, and from about 0.5 to 5% of amixture Of C₈ to C₁₄ fatty acids. Other constituents of the presentinvention may include water, an alcohol (such as ethanol or propanol) ora mixture thereof.

[0021] Also disclosed is a novel method of killing harmful microbes onthe udder of milk-producing animal using the above-mentionedcomposition.

[0022] Accordingly, it is an object of the present invention to providean effective antimicrobial potent enough to kill a broad-spectrum ofharmful microorganisms.

[0023] It is another object of the invention to provide an effectiveantimicrobial which is non-irritating upon application.

[0024] It is a further object of the invention to provide an effectiveantimicrobial that is non-toxic to animals and to humans working withthe animals.

[0025] It is an additional object of the invention to provide aneffective antimicrobial that does not pose a significant health-risk asa residual in the milk produced by the animals to which it is applied.

[0026] It is still another object of the invention to provide aneffective antimicrobial that is stable over time and with variations inconcentration/contaminants, and provides an enhanced residual effectwhen applied to a teat or udder.

DISCLOSURE OF THE PREFERRED EMBODIMENTS

[0027] The preferred embodiment of the present invention is a solutionof 70% by weight to propylene glycol as the primary solvent and 1.0% byweight of a mixture of C₈ and C₁₀ aliphatic fatty acids as the solute.The fatty acid mixture preferably includes 55% by weight C₈ fatty acidand 40% by weight CIO fatty acids, with the remaining 5% by weight ofthe fatty acids mixture being made up of other short to medium-chainfatty acids. The remaining 29% by weight of the composition includeswater as a secondary solvent, with trace concentrations of compatibleperfumes and dyes. Additionally, a preservative such as methyl parabenor propyl paraben may be added to increase the life of the composition.These constituents are combined to form a solution using methods knownto one of ordinary skill in the art.

[0028] The present invention is not limited to the composition of thepreferred embodiment, but may contain a variety of primary and secondarysolvents and solutes in accordance with the teachings of thisdisclosure. The concentrations of solvents and solutes may also bevaried while remaining within the scope of the present invention.

[0029] For example, the primary solvent may be selected from the groupof lipophilic polar solvents including propylene glycol, ethylen glycol,glycol, and isopropanol. The secondary solvent may include water, analcohol (such as ethanol or propanol) or a mixture thereof.

[0030] Preferably, the mixture of fatty acids is a combination of C₈ andC₁₀ aliphatic fatty acids. However, mixtures of short to medium-chainaliphatic fatty acids, such as C₈, C₁₀, C₁₂ and C₁₄ aliphatic fattyacids, are encompassed by the present invention.

[0031] Additionally, the concentration of the primary solvent may varyfrom about 60 to 95% by weight of the composition. The concentration ofthe solute may also vary from about 0.5 to 5% of the composition, andthe C₈ and C₁₀ fatty acids in the solute may also vary about thepreferred concentrations of 55% and 40%, respectively.

[0032] Formulations of the preferred embodiments have demonstratedimpressive antimicrobial efficacy against a broad spectrum ofmastitis-causing microorganisms, both in vitro in the laboratory and invivo during testing with dairy cows. The formulations of the preferredembodiments have been shown to have germicidal efficacy comparable tosuch popular chemical germicides as the iodophors, chlorhexidine-basedcompounds and chlorine-based compounds. Additionally, the mixture offatty acids with a lipophilic polar solvent as disclosed has been shownto be superior in action to compositions wherein a lipophilic polarsolvent and a single length fatty acid is used.

EXAMPLE 1

[0033] A set of in vitro tests was run on Staphylococcus aureus,Escherichia coli, Streptococcus agalactiae, Streptococcus uberis,Staphylococcus epidermidis, and Pseudomonas aeruginosa. A culture of themicroorganism was first prepared according to the following procedure.Six to ten milliliters of trypticase soy broth was inoculated with theorganism under consideration from a frozen or refrigerated stock. Afterincubation for five to seven hours at 37° C., the culture wastransferred to a flask containing trypticase soy broth. The culture inthe flask was then incubated on a gyratory shaker at approximately 1500rpm overnight at 37° C. The next day, the culture in the flask is washedonce in phosphate buffered saline solution (PBS) and stored atapproximately 4° C. until needed.

[0034] To test a specific antimicrobial's efficacy, ten milliliters ofthe culture was transferred to a 15 milliliter centrifuge tube andcentrifuged at approximately 600 to 700 g for approximately five to tenminutes. After decanting the supernatant, ten milliliters of theantimicrobial (or PBS in the negative, or untreated, control) was added,and the centrifuge tube vortexed to suspend the bacteria. In those testsinvolving “organic loads”, a 1:1 dilution of the antimicrobial withwhole milk was made before addition to the cultured microorganism tosimulate conditions the product may have to experience in a real lifeenvironment, such as on a cow's teat.

[0035] The tube was then placed in a 37° C. water bath for 1 minute. Thetube was centrifuged again at approximately 600 to 700 g forapproximately five to ten minutes, and the supernatant decanted. Theremaining precipitate was then suspended in a quencher solutioncontaining letheen broth, 1% sodium thiosulfate and 0.2 mM CaCl₂. Thetube was centrifuged at approximately 600 to 700 g for approximatelyfive to ten minutes, decanted and washed a second time in the quenchersolution.

[0036] The surviving bacteria suspended in quencher solution were thendiluted with PBS and plated on appropriate media, for example, MacConkeyfor Escherichia coli, Staph 110 for Staphylococcus, and trypticase soyagar with 6% Sheep's blood for Streptococcus. The plates were incubated24 to 48 hours at 37° C. and counted. The surviving organisms wereexpressed as colony forming units (CFU)/ml, and compared to the negativecontrol as percent survival.

Staphylococcus Aureus Undiluted Antimicrobial

[0037] Antimicrobial Tested Percent Survival 93% propylene glycol/7%0.003 water 1% fatty acid mixture^(a)/93% 0 propylene glycol/6% water 1%fatty acid mixture/70% 0 propylene glycol/29% water 0.5% iodophor 0 1%iodophor 0 0.5% chlorhexidine 0 Chlorine dioxide 0

Diluted with Organic Load

[0038] Antimicrobial tested Percent Survival 93% propylene glycol/7%30.83 water 70% propylene 94.23 glycol/30% water 0.25% fatty acid 27.86mixture/93% propylene glycol/6.75% water 0.5% fatty acid 0 mixture/93%propylene glycol/6.5% 1% fatty acid 0 mixture/99% propylene glycol 1%fatty acid 5.18 mixture/93% propylene glycol/6% water 1% fatty acid 3.65mixture/70% propylene glycol/29% water 1% fatty acid 0 mixture/60%propylene glycol/39% water 2% fatty acid 0 mixture/93% propyleneglycol/5% water 3% fatty acid 0 mixture/93% propylene glycol/4% water 4%fatty acid 0 mixture/93% propylene glycol/3% water 5% fatty acid 0mixture/93% propylene glycol/2% water 1% fatty acid 0 mixture/70%propylene glycol/25% propanol/4% water 1% fatty acid 3.36mixture^(b)/70% propylene glycol/29% water 0.5% iodophor 0.70 1%iodophor 0.003 0.5% chlorhexidine 0.345 chlorine dioxide 16.96

Escherichia Coli Undiluted Antimicrobial

[0039] Antimicrobial tested Percent Survival 93% propylene glycol/7% 0water 1% fatty acid 0 mixture/93% propylene glycol/6% water 1% fattyacid 0 mixture/70% propylene glycol/29% water 0.5% iodophor 0 1%iodophor 0 0.5% chlorhexidine 0 chlorine dioxide 0

Diluted with Organic Load

[0040] Antimicrobial tested Percent Survival 93% propylene glycol/7%2.43 water 0.25% fatty acid 7.64 mixture/93% propylene glycol/6.75%water 0.5% fatty acid 0 mixture/93% propylene glycol/6.5% water 1% fattyacid 0 mixture/99% propylene glycol 1% fatty acid 0 mixture/93%propylene glycol/6% water 1% fatty acid 0 mixture/70% propyleneglycol/29% water 1% fatty acid 0 mixture/60% propylene glycol/39% water2% fatty acid 0 mixture/93% propylene glycol/5% water 3% fatty acid 0mixture/93% propylene glycol/4% water 4% fatty acid 0 mixture/93%propylene glycol/3% water 5% fatty acid 0 mixture/93% propyleneglycol/2% water 1% fatty acid 0 mixture/70% propylene glycol/25%propanol/4% water 1% fatty acid 0 mixture^(b)/70% propylene glycol/29%water 0.5% iodophor 0.003 1% iodophor 0 0.5% chlorhexidine 0 chlorinedioxide 0

Streptococcus Agalactiae Diluted with Organic Load

[0041] Antimicrobial tested Percent Survival 1% fatty acid 0 mixture/70%propylene glycol/29% water 0.5% iodophor 0 1% iodophor 0 0.5%chlorhexidine 0 chlorine dioxide 0

Streptococcus Uberis Diluted with Organic Load

[0042] Antimicrobial tested Percent Survival 1% fatty acid 0.08mixture/70% propylene glycol/29% water 0.5% iodophor 0.01 1.0% iodophor0 0.5% chlorhexidine 0.01 chlorine dioxide 5.19

Staphylococcus Epidermidis Diluted with Organic Load

[0043] Antimicrobial tested Percent Survival 1% fatty acid 0 mixture/70%propylene glycol/29% water 0.5% iodophor 0 1% iodophor 0 0.5% iodophor 0chlorine dioxide 5

Pseudomonoas Aeruginosa Diluted with Organic Load

[0044] Antimicrobial tested Percent Survival 1% fatty acid 0 mixture/70%propylene glycol/29% water 0.5% iodophor 0 1% iodophor 0 0.5%chlorhexidine 28.98 chlorine dioxide 0

[0045] The results of in vitro tests performed in Example 1 indicatethat an antimicrobial including varying concentrations of a mixture offatty acids, propylene glycol and water are as efficacious as, if notmore efficacious than, chemical compositions presently in use as teatdip antimicrobials. Additionally, these tests confirm thatantimicrobials containing different mixtures of short to medium-chainfatty acids are as efficacious, if not more efficacious than, chemicalcompositions presently in use as teat dip antimicrobials.

EXAMPLE 2

[0046] Another set of in vitro tests were run on Staphylococcus aureus,Escherichia coli, and Streptococcus uberis. A culture of themicroorganism was first prepared according to the following procedure.Six to ten milliliters of trypticase soy broth was inoculated with theorganism under consideration from a frozen or refrigerated stock. Afterincubation for five to seven hours at 37° C., the culture wastransferred to a flask containing 200 milliliters of trypticase soybroth. The culture in the flask was then incubated on a gyratory shakerat approximately 1500 rpm overnight at 37° C. The next day, the culturein the flask is washed once in PBS or 0.1% protease peptone, and storedat approximately 4° C. until required.

[0047] The tests were run using dairy cow teats which had been obtainedfrom a slaughter house. The teats were cut off at their connection tothe udder, cleaned, and all hair was removed by using an open flame. Theteats selected for the testing were screened for lesions, cuts, scars orother abnormalities. The teats were stored until needed in plastic bagsin ice at a temperature below freezing.

[0048] Immediately before the test, the teats were thawed in warm waterand thoroughly dried with a paper towel. The thawed teats were suspendedfrom clips attached to a horizontal rod. Prior to testing with theselected organism, the teats were disinfected by dipping the teat in asolution of 70% propanol and air-drying the teat.

[0049] The microorganism in question was placed in a 50 milliliterbeaker for testing purposes. Each teat was dipped into culture and leftto drain for five minutes. The teats were then dipped into a beakercontaining the selected antimicrobial (or sterile water if control) andleft to drain for ten minutes. Ten teats were tested per antimicrobial(or control).

[0050] The teats were then dipped into quencher solution to recover thesurviving organisms. The quencher solution included Bacto-Letheen brothand 1% sodium thiosulfate.

[0051] The beakers containing the quencher solution and teat weresuspended in an ultrasonic water bath and sonicated for 30 seconds. Thesolutions containing the surviving organisms were transferred to 25×150millimeter tubes, vortexed, and diluted with PBS or 1% protease peptone.

[0052] Countable dilutions were plated on appropriate media, andincubated from 24 to 48 hours at 37° C. The colonies were countedmacroscopically with the aid of fluorescent light. Non-typical colonieswere not counted. The results were expressed in CFU/ml, the log of themean value of each antimicrobial was taken. The log values for theantimicrobials were then compared with the log value for the negativecontrol to obtain a percent log reduction.

Staphylococcus Aureus

[0053] Antimicrobial tested Percent Log Reduction 1% fatty acid 36.20mixture/93% propylene glycol/6% water 0.5% iodophor 26.72 1% iodophor46.35 chlorine dioxide 33.90

Escherichia Coli

[0054] Antimicrobial tested Percent Log Reduction 1% fatty acid 37.80mixture/93% propylene glycol/6% water 0.5% iodophor 32.38 1% iodophor45.17 0.5% chlorhexidine 20.50 chlorine dioxide 22

Streptococcus Uberis

[0055] Antimicrobial tested Percent Log Reduction 1% fatty acid 29.5mixture/93% propylene glycol/6% water 1% iodophor 22.47

[0056] The results of in vitro tests performed in Example 2 indicatethat antimicrobials including fatty acids, propylene glycol and waterare as efficacious as, if not more efficacious than, chemicalcompositions presently in use as teat dip antimicrobials.

EXAMPLE 3

[0057] A set of in vivo tests was run on Staphylococcus aureus andStreptococcus agalactiae using one hundred and fifty Holstein cows. Thecows were housed in free stalls with concrete surfaces, bedded with dryshavings, and milked in a herringbone parlor.

[0058] The cows were screened for pre-existing infections ofStaphylococcus aureus, Streptococcus agalactiae, and Streptococcusuberis, and for injuries on the teats. Cows experiencing other types ofinfections were divided equally among the test groups.

[0059] The control group was not treated with either a pre- orpost-milking antimicrobial. A second group was treated with both a pre-and post-milking iodophor antimicrobial (0.5% iodophor). A third groupwas treated with another pre- and post-milking iodophor antimicrobial(1.0% iodophor). The remaining cows were treated before and/or aftermilking with a preferred embodiment of the present invention.

[0060] Where no pre-milking antimicrobial was used, the udder wasforestripped, washed, and dried with a paper towel before milking. Foranimals receiving both pre- and post-milking antimicrobial applications,the udder was forestripped prior to a 20 to 30 second pre-milkingapplication of antimicrobial, and then manually dried with a papertowel.

[0061] The milking machines were attached with three minutes after udderpreparation, and removed by automatic detaching devices.

[0062] All teats were exposed to the microbe culture equally by dippingthe teat in a suspension of the test microbe to a depth of approximately25 mm immediately after the afternoon milking. After exposure to theculture, the teats were dipped immediately in the antimicrobial underconsideration.

[0063] Duplicate milk samples from each quarter of udder of the testanimals were collected and cultured weekly. Additional milk samples werecollected from animals where the results from the first two samplesdiffered. The results of the tests were expressed as a percentreduction.

Staphylococcus Aureaus

[0064] Antimicrobial tested Percent Reduction 1% fatty acid 78.60mixture/93% propylene glycol/6% water 0.5% iodophor 84.0 1% iodophor85.30

Streptococcus Agalactiae

[0065] Antimicrobial tested Percent Reduction 1% fatty acid 60.30mixture/93% propylene glycol/6% water 0.5% iodophor 77.30 1% iodophor79.20

[0066] The results of in vivo tests performed in Example 3 indicate thatantimicrobials including fatty acids, propylene glycol and water are asefficacious as chemical compositions presently in use as teat dipantimicrobials.

EXAMPLE 4

[0067] In a further set of in vitro tests, the antimicrobial efficacy ofan antimicrobial solution including a mixture of short to medium-chainfatty acids dissolved in propylene glycol was compared with anantimicrobial solution including a single length fatty acid dissolved inpropylene glycol. The selected test culture was Staphylococcus aureus,and the procedure followed was that outlined above in Example 1.

Diluted with Organic Load

[0068] Antimicrobial tested CFU/ml Control  6.5 × 10⁸ 1% fatty acid 70mixture/99% propylene glycol 1% C₁₀ fatty acid/99% 4.03 × 10³ propyleneglycol

[0069] The test performed in Example 4 confirm that a significantdifference in antimicrobial effect occurs when a mixture of short tomedium-chain fatty acids are used in comparison with the use of a singlelength fatty acid.

[0070] In part, the impressive antimicrobial effect of the presentinvention is thought to be caused by the mixture of short tomedium-chain fatty acids. In fact, it has been theorized that the longerchain molecules (C₁₀ to C₁₂), such as are used in the compositionsdisclosed herein, have a significant efficacy against certain bacteria,especially gram positives.

[0071] Additionally, propylene glycol also has a measurableantimicrobial effect, as can be seen in the results presented in Example1, and as recognized by others in the art. It has been suggested thatcertain species, and strains within species of bacteria, areparticularly susceptible to the bactericidal effects of propyleneglycol. See P. A. Thomas et al., Antibacterial Properties of DiluteFormocresol and Eugenol and Propylene Glycol, Oral Surgery, February1980 and I. Olitzky, Antimicrobial Properties of a Propylene GlycolBased Topical Therapeutic Agent, Journal of Pharmaceutical Sciences, May1965.

[0072] However, the results also show an increased antimicrobialresponse when the fatty acids are combined with propylene glycol, orothers of the solvents listed above. Although not intending to be boundby any theory, it is believed, that the degree of increased bactericidalefficacy of the fatty acids is determined to a large degree by thepolarity of the solvent. It is further believed that the polarity ofpropylene glycol, or others of the solvents listed above, is uniquelysuited to trigger a synergistic reaction between the solvent and thesolute.

[0073] Polarity or conductivity of a material is a measurement of thedielectric constant, the ratio of electric displacement to electricfield intensity. At one extreme, is a vehicle such as water, with a veryhigh polarity, and dielectric constant of 88. At the other extreme,petroleum and vegetables oils (such as olive or cottonseed oil) have avery low polarity, with a dielectric constant of about 2. Propyleneglycol is moderately polar, with a dielectric constant of about 41. Bycomparison, low molecular weight alcohols have lower polarities, withdielectric constants in the range of 15 to 25.

[0074] It is believed that a greater degree of antimicrobial efficacyoccurs when the fatty acids are solubilized in lipophilic high polaritysolvents versus when they are solubilized in lipophilic low polaritysolvents. When 1% by weight of a mixture of 55% C₈ and 40% C₁₀ fattyacids is added to lipophilic, high polarity solvents such as propyleneglycol, glycerol, ethylene glycol or isopropanol, the bactericidalefficacy against such organisms as Staphylococcus aureus and Escherichiacoli is impressive. However, when the same concentration of fatty acidsis solubilized in such low polarity lipophilic solvents such as corn oilor mineral oil, the bactericidal efficacy of the fatty acid componentdecreases significantly, with a large number of the organisms survivingthe treatment.

EXAMPLE 5

[0075] In a further set of in vitro tests, the antimicrobial efficacy ofa fatty acid antimicrobial in solvents of various polarities was tested.The selected test cultures were Staphylococcus aureus and Escherichiacoli, and the procedure followed was that outlined above in Example 1.

Staphylococcus AureusDiluted with Organic Load

[0076] Vehicle Dielectric constant Percent Survival Corn oil 2-4 41.5Mineral oil 2-4 100 Propylene 41 5.2^(a) Glycol Ethylene Glycol 410.5^(a) Glycerol 47 14.86^(a) Water^(b) 88 0

Escherichia ColiDiluted with Organic Load

[0077] Dielectric Percent Vehicle Constant Survival Corn oil 2-4   56.18Mineral oil 2-4    6.86 Propylene 41   0^(a) Glycol Ethylene Glycol 41  0^(a) Glycerol 47   0^(a) Water^(b) 88  0

[0078] The results of the in vitro tests performed in Example 5 indicatethat an antimicrobial solution of fatty acids in solution with propyleneglycol, ethylene glycol or glycerol, or solubilized in water, areeffective antimicrobials, whereas solutions using corn oil and mineraloil are generally less effective antimicrobials.

[0079] The exact theory behind the synergistic reaction between themixtures of fatty acids and the lipophilic polar solvents is as of yetunknown. However, Corner reported a similar synergistic response when C₈fatty acids were added to fatty alcohols such as octanol. Cornerconcluded that the fatty acids interact with lipophilic companionmolecules, such as alcohols, so as to shield the charges on the polarend of the fatty acid molecule. The shielding is thought to protect theproton shuttle mechanism that is responsible for uncoupling thetransmembrane proton gradients in the membrane of bacterial cells which,in turn, inhibits that cells' ability to grow. Corner also hypothesizedthat the fatty acids helped disperse alcohol micelle aggregates whichallowed the alcohol to have better contact with cells, therebyincreasing its ability to kill more cells.

[0080] It cannot be concluded, however, based on the tests run to date,whether the propylene glycol is indeed aiding the fatty acids astheorized by Corner above, or if the acids are aiding the propyleneglycol in some yet unknown fashion, or if there is a mutual synergismoccurring. Empirical data is not available in the laboratory or in theliterature that would fully explain the synergism observed.

[0081] Testing has confirmed that the preferred embodiments of thepresent invention are non-irritating to the skin. This characteristic ofthe present invention has been demonstrated in vivo using the standarddermal irritation test with rabbits, commonly referred to as the Draizeskin test. These results were confirmed using high sensitivity dermalresponse in vitro technology, namely the two-part system (including anartificial membrane-dye and a macromolecular matrix) manufactured byIn-Vitro International and sold as the “IRRITECTION” Assay System or the“SKINTEX” Assay System.

[0082] In fact, it is believed that the high percentage of glycol in thepreferred embodiments enhances this invention's non-irritatingcharacteristics, and may actually help protect and heal the skin.Glycols, such as propylene glycol, are widely known as humectants withvery good skin protecting properties.

[0083] It should also be noted that the components of this product arealso non-toxic. The fatty acids (at the low concentrations proposed) andpropylene glycol are both considered safe, with propylene glycol beingrecognized as a good grade material by the Food and Drug Administration.There is no evidence that the concentrations of fatty acids or propyleneglycol present in the scope of the present invention are harmful toanimals, humans or the environment.

[0084] Additionally, the preferred embodiments of the present inventionhave superior residual activity to those chemical germicides presentlybeing used as teat dips, washes, sprays and wipes. Given the solubilityof fatty acids in propylene glycol, the concentrations of fatty acidsand propylene glycol are known to be stable over time even withvariations in such conditions as temperature, humidity, andcontamination with milk. Moreover, because of the high percentage ofpropylene glycol, this product has a relatively high viscosity, beingconsiderably higher than that of water and many other products utilizedtoday in the dairy industry as post-milking teat dips. The higherviscosity gives this product the ability to stay on the teat longer at ahigher concentration.

[0085] Still other aspects, objects and advantages of the presentinvention can be obtained from a study of the specification and theappended claims.

1. A method for reducing the incidence of mastitis in a dairy animal,the method comprising the step of: topically applying an antimicrobialcomposition to the teats of the animal, the composition comprising (1)from about 60% to about 95% of a lipophilic polar solvent selected fromthe group consisting of propylene glycol, ethylene glycol, glycerol,isopropanol, and sorbitol, by weight of the composition, (2) at leasttwo C₈ to C₁₄ fatty acids in a total amount of from about 0.5% to about5% by weight of the composition; and (3) devoid of sufficient fatty acidester to substantially improve the antimicrobial activity of thecomposition.
 2. The method of claim 1, where the lipophilic polarsolvent is propylene glycol.
 3. The method of claim 1, where thelipophilic polar solvent is present in an amount from about 60% to about75% by weight of the composition.
 4. A method for reducing the incidenceof mastitis in a dairy animal, the method comprising the step of:topically applying an antimicrobial composition to the teats or udder ofthe animal, the composition comprising: from about 50% to about 99% of alipophilic polar solvent selected from the group consisting of propyleneglycol, ethylene glycol, glycerol, isopropanol, and sorbitol, by weightof composition; a C₈ to C₁₄ fatty acid in the total amount from about0.5% to 5% by weight of the composition; and devoid of sufficient fattyacid ester to substantially improve the antimicrobial activity of thecomposition.
 5. The method of claim 4 wherein the composition furthercomprises a second C₈ to C₁₄ fatty acid.
 6. The method of claim 4,wherein the lipophilic polar solvent is propylene glycol.
 7. The methodof claim 4 wherein the lipophilic polar solvent is present in the amountfrom about 50% to about 75% by weight of composition.
 8. The method ofclaim 4 wherein the lipophilic polar solvent is present in the amountfrom about 60% to about 99% by weight of composition.
 9. The method ofclaim 4 wherein the fatty acid is caprylic acid.
 10. The method of claim4 wherein the fatty acid is capric acid.
 11. A method for reducing theincidence of mastitis in a dairy animal, the method comprising the stepof: topically applying an antimicrobial composition to the teats of theanimal, the composition comprising; from about 50% to 99% by weight ofthe composition a lipophilic polar solvent having a dielectric constantgreater than 25, a C₈ to C₁₄ fatty acid in the total amount of fromabout 0.5% to about 5% by weight of the composition, and devoid ofsufficient fatty acid ester to substantially improve the antimicrobialactivity of the composition.
 12. The method of claim 11, wherein thelipophilic polar solvent is selected from a group consisting ofpropylene glycol, ethylene, glycol, glycerol, isopropanol, and sorbitol.13. The method of claim 11, wherein the antimicrobial compositionfurther comprises a second C₈ to C₁₄ fatty acid.
 14. The method of claim11, wherein the antimicrobial composition has a pH below about
 4. 15.The method of claim 11, wherein the antimicrobial composition furthercomprises a second C₁₂ or C₁₄ fatty acid.
 16. The method of claim 11,wherein the composition further comprises a C₇ fatty acid.
 17. A methodfor reducing the incidence of mastitis in a dairy animal, the methodcomprising the step of: topically applying an antimicrobial compositionto the teats or udder of the animal, the composition comprising: fromabout 50% to about 99% of a lipophilic polar solvent having a dielectricconstant greater than 25 by weight of composition; a C₇ to C₁₄ fattyacid in the total amount from about 0.5% to 5% by weight of thecomposition; and devoid of sufficient fatty acid ester to substantiallyimprove the antimicrobial activity of the composition.
 18. The method ofclaim 17 wherein the composition further comprises a second C₇ to C₁₄fatty acid.
 19. The method of claim 17, wherein the lipophilic polarsolvent is selected from the group consisting of propylene glycol,ethylene glycol, glycerol, isopropanol, and sorbitol.
 20. The method ofclaim 17 wherein the lipophilic polar solvent is present in the amountfrom about 50% to about 75% by weight of composition.
 21. The method ofclaim 17 wherein the lipophilic polar solvent is present in the amountfrom about 60% to about 99% by weight of composition.
 22. The method ofclaim 17 wherein the fatty acid is caprylic acid.
 23. The method ofclaim 17 wherein the fatty acid is capric acid.
 24. An antimicrobialcomposition for reducing the incidence of mastitis in a dairy animal,the composition comprising: from about 50% to about 99% of a lipophilicpolar solvent having a dielectric constant greater than 25, by weight ofthe composition; a C₇ to C₁₄ fatty acid in the total amount of fromabout 0.5% to about 5% by weight of the composition; and devoid ofsufficient fatty acid ester to substantially improve the antimicrobialactivity of the composition.
 25. The antimicrobial composition of claim24, wherein the lipophilic polar solvent is selected from a groupconsisting of: propylene glycol, ethylene glycol, glycerol, isopropanol,and sorbitol.
 26. The antimicrobial composition of claim 24, wherein thecomposition further comprises a second C₇ to C₁₄ fatty acid.
 27. Theantimicrobial composition of claim 24, wherein the antimicrobialcomposition has a pH below about
 4. 28. The antimicrobial composition ofclaim 24, wherein the fatty acid mixture consists essentially of C₇, C₈,C₉, C₁₀, C₁₂ and C₁₄ fatty acids.